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© 2022 The Authors; Published by the American Association for Cancer Research. This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 International (CC BY-NC-ND), Purpose: Cetuximab is an EGFR-targeted therapy approved for the treatment of RAS wild-type (WT) metastatic colorectal cancer (mCRC). However, about 60% of these patients show innate resistance to cetuximab. To increase cetuximab efficacy, it is crucial to successfully identify responder patients, as well as to develop new therapeutic approaches to overcome cetuximab resistance. Experimental design: We evaluated the value of EGFR effector phospholipase C gamma 1 (PLCγ1) in predicting cetuximab responses, by analyzing progression-free survival (PFS) of a multicentric retrospective cohort of 94 treated patients with mCRC (log-rank test and Cox regression model). Furthermore, we used in vitro and zebrafish xenotransplant models to identify and target the mechanism behind PLCγ1-mediated resistance to cetuximab. Results: In this study, levels of PLCγ1 were found increased in RAS WT tumors and were able to predict cetuximab responses in clinical samples and in vitro and in vivo models. Mechanistically, PLCγ1 expression was found to bypass cetuximab-dependent EGFR inhibition by activating ERK and AKT pathways. This novel resistance mechanism involves a noncatalytic role of PLCγ1 SH2 tandem domains in the propagation of downstream signaling via SH2-containing protein tyrosine phosphatase 2 (SHP2). Accordingly, SHP2 inhibition sensitizes PLCγ1-resistant cells to cetuximab. Conclusions: Our discoveries reveal the potential of PLCγ1 as a predictive biomarker for cetuximab responses and suggest an alternative therapeutic approach to circumvent PLCγ1-mediated resistance to cetuximab in patients with RAS WT mCRC. In this way, this work contributes to the development of novel strategies in the medical management and treatment of patients with mCRC., M. Martins' research was supported by Liga Portuguesa Contra o Cancro (LPCC): Terry Fox Fundation; Investigador FCT- Fundação para a Ciência e Technologia (IF/00409/2014) and IMM Bridge grant; RC-D research was supported by Fundação para a Ciência e Technologia (SFRH/BD/139138/2018). A. Fernandes was supported by LPCC-IMM BIOBANK; R. Fior was supported by Champalimaud Foundation and L. Costa was supported by Merck Serono.

Purpose:Cetuximab is an EGFR-targeted therapy approved for the treatment of RAS wild-type (WT) metastatic colorectal cancer (mCRC). However, about 60% of these patients show innate resistance to cetuximab. To increase cetuximab efficacy, it is crucial to successfully identify responder patients, as well as to develop new therapeutic approaches to overcome cetuximab resistance.Experimental Design:We evaluated the value of EGFR effector phospholipase C gamma 1 (PLCγ1) in predicting cetuximab responses, by analyzing progression-free survival (PFS) of a multicentric retrospective cohort of 94 treated patients with mCRC (log-rank test and Cox regression model). Furthermore, we used in vitro and zebrafish xenotransplant models to identify and target the mechanism behind PLCγ1-mediated resistance to cetuximab.Results:In this study, levels of PLCγ1 were found increased in RAS WT tumors and were able to predict cetuximab responses in clinical samples and in vitro and in vivo models. Mechanistically, PLCγ1 expression was found to bypass cetuximab-dependent EGFR inhibition by activating ERK and AKT pathways. This novel resistance mechanism involves a noncatalytic role of PLCγ1 SH2 tandem domains in the propagation of downstream signaling via SH2-containing protein tyrosine phosphatase 2 (SHP2). Accordingly, SHP2 inhibition sensitizes PLCγ1-resistant cells to cetuximab.Conclusions:Our discoveries reveal the potential of PLCγ1 as a predictive biomarker for cetuximab responses and suggest an alternative therapeutic approach to circumvent PLCγ1-mediated resistance to cetuximab in patients with RAS WT mCRC. In this way, this work contributes to the development of novel strategies in the medical management and treatment of patients with mCRC.

Supplementary Figure from Predictive and Therapeutic Implications of a Novel PLCγ1/SHP2-Driven Mechanism of Cetuximab Resistance in Metastatic Colorectal Cancer

Supplementary Data from Predictive and Therapeutic Implications of a Novel PLCγ1/SHP2-Driven Mechanism of Cetuximab Resistance in Metastatic Colorectal Cancer

Most cancers reactivate telomerase to maintain telomere length to acquire immortality. The importance of this process is well illustrated by the fact that telomerase promoter mutations are found at a high frequency in many cancer types, including melanoma. However, it is unclear when and if telomerase is strictly required during tumorigenesis. Here, we show that melanoma can occur in the absence of telomerase but is required to sustain later growth and to avoid tumor regression. We combined telomerase mutant zebrafish (tert-/-) with two established melanoma models and found equal melanoma incidence and invasiveness as tumors became visible. Later, however, whiletert+/+fish develop increasing larger tumors,tert-/-tumors stagnate growth and regress.tert-/-tumors showed lower cell proliferation, higher apoptosis and melanocyte differentiation. We also detected an immune response directed attert-/-tumors.tert-/-tumors exhibited increased immune cell infiltrates and resume growth when transplanted into immunocompromised hosts. We propose that telomerase is required for melanoma in zebrafish, albeit at later stages of progression, to sustain growth while avoiding immune rejection and regression. Thus, absence of telomerase restricts melanoma through tumor-autonomous mechanisms (cell cycle arrest, apoptosis and melanocyte differentiation) and a non-tumor-autonomous mechanisms (immune rejection).

Telomeres are nucleoprotein complexes that protect the chromosome-ends from eliciting DNA repair while ensuring their complete duplication. Pot1 is a subunit of telomere capping complex that binds to the G-rich overhang and inhibits the activation of DNA damage checkpoints. In this study, we explore new functions of fission yeast Pot1 by using apot1-1temperature sensitive mutant. We show thatpot1inactivation impairs telomere DNA replication resulting in the accumulation of ssDNA leading to the complete loss of telomeric DNA. Recruitment of Stn1 to telomeres, an auxiliary factor of DNA lagging strand synthesis, is reduced inpot1-1mutants and overexpression of Stn1 rescues loss of telomeres and cell viability at restrictive temperature. We propose that Pot1 plays a crucial function in telomere DNA replication by recruiting Stn1-Ten1 and Polα-primase complex to telomeres, thus promoting lagging-strand DNA synthesis at stalled replication forks.

Telomere shortening is a hallmark of aging and is counteracted by telomerase. As in humans, the zebrafish gut is one of the organs with the fastest rate of telomere decline, triggering early tissue dysfunction during normal zebrafish aging and in prematurely aged telomerase mutants. However, whether telomere-dependent aging of an individual organ, the gut, causes systemic aging is unknown. Here we show that tissue-specific telomerase expression in the gut can prevent telomere shortening and rescues premature aging of tert−/−. Induction of telomerase rescues gut senescence and low cell proliferation, while restoring tissue integrity, inflammation and age-dependent microbiota dysbiosis. Averting gut aging causes systemic beneficial impacts, rescuing aging of distant organs such as reproductive and hematopoietic systems. Conclusively, we show that gut-specific telomerase expression extends the lifespan of tert−/− by 40%, while ameliorating natural aging. Our work demonstrates that gut-specific rescue of telomerase expression leading to telomere elongation is sufficient to systemically counteract aging in zebrafish.

Telomeres and telomerase prevent the continuous erosion of chromosome-ends caused by lifelong cell division. Shortened telomeres are associated with age-related pathologies. While short telomere length is positively correlated with increased lethality at the individual level, in comparisons across species short telomeres are associated with long (and not short) lifespans. Here, we tested this contradiction between individual and evolutionary patterns in telomere length using African annual killifish. We analysed lifespan and telomere length in a set of captive strains derived from well-defined wild populations of Nothobranchius furzeri and its sister species, N. kadleci, from sites along a strong gradient of aridity which ultimately determines maximum natural lifespan. Overall, males were shorter-lived than females, and also had shorter telomeres. Male lifespan (measured in controlled laboratory conditions) was positively associated with the amount of annual rainfall in the site of strain origin. However, fish from wetter climates had shorter telomeres. In addition, individual fish which grew largest over the juvenile period possessed shorter telomeres at the onset of adulthood. This demonstrates that individual condition and environmentally-driven selection indeed modulate the relationship between telomere length and lifespan in opposite directions, validating the existence of inverse trends within a single taxon. Intra-individual heterogeneity of telomere length (capable to detect very short telomeres) was not associated with mean telomere length, suggesting that the shortest telomeres are controlled by regulatory pathways other than those that determine mean telomere length. The substantial variation in telomere length between strains from different environments identifies killifish as a powerful system in understanding the adaptive value of telomere length.

Telomere shortening is a hallmark of aging and is counteracted by telomerase. The gut is one of the earliest organs to exhibit short telomeres and tissue dysfunction during normal zebrafish aging. This is recapitulated in prematurely aged telomerase mutants (tert-/-). Here, we show that gut-specific telomerase activity in tert-/- zebrafish prevents premature aging. Induction of telomerase rescues gut senescence and low cell proliferation to wild-type levels, while restoring gut tissue integrity, inflammation, and age-dependent gut microbiota dysbiosis. Remarkably, averting gut dysfunction results in a systemic beneficial impact. Gut-specific telomerase activity rescues premature aging markers in remote organs, such as the reproductive (testes) and hematopoietic (kidney marrow) systems. Functionally, it also rescues age-dependent loss of male fertility and testes atrophy. Finally, we show that gut-specific telomerase activity increases the lifespan of telomerase mutants. Our work demonstrates that delaying telomere shortening in the gut is sufficient to systemically counteract aging in zebrafish.

Tumor models allowing for the in vivo investigation of molecular mechanisms driving tumor progression and metastasis are important to develop novel strategies for cancer treatment. Unfortunately, for Ewing sarcoma no adequate genetic animal models are currently available. Mouse xenograft models are the state of the art to model Ewing sarcoma in vivo. Here, we describe an alternative Ewing sarcoma xenograft model in embryonic and larval zebrafish. This xenograft model offers live imaging and easy compound testing opportunities hereby complementing mouse xenograft models. In this chapter, we provide a detailed protocol how to xenograft Ewing sarcoma cells (shSK-E17T) into 2-day-old zebrafish and how xenografted zebrafish can be imaged and analyzed over consecutive days to study tumor proliferation.

Background: VEGF-A is the most potent pro-angiogenic factor and is upregulated in a variety of tumors. Therapies were developed to neutralize VEGF signalling, such as Bevacizumab (Bev). Despite promising pre-clinical results, the average response rate to Bev is reduced for most cancers, with significant side effects. In breast cancer (BC) several studies and a meta-analysis have also confirmed a limited i efficacy in unselected populations. Lack of predictive biomarkers prevents a good patient selection. As such guidelines consider Bev only in a minority of cases. Additionally, depending on the individual tumor, VEGF-A can either block or promote metastasis. In this context, an assay able to predict individual responses to Bev, including its impact on metastasis would prove of great value. Methods: We developed an assay, using zebrafish xenografts(zX), where tumor response and the impact on angiogenesis and metastasis (mets) can be readily analysed. By using Triple negative BC (TNBC) cell line models (Hs578T and MDA-MB-468) and zebrafish patient derived xenografts(zPDX) we evaluated the impact of Bev on proliferation, apoptotis, tumor size, angiogenesis and metastasis. Results: a) Human TNBC cell lines display different tumoral features in zX: Hs578T presented the most vascularized tumors (vessel density ~32.4%) and the lowest metastatic capacity (2.6% of xenografts presented mets, n=77). In contrast, MDA-MB-468 that only recruits blood vessels to the base of the tumor, showed a higher metastatic potential (42.5%, n=87). No direct correlation between vessel density and metastasis could be seen. b) zX were able to reveal different individual tumor responses to Bev in 4 days. Analysis of cell death revealed higher apoptosis rate in Hs578T xenografts (1.6-fold, p Conclusions: Our results show that the zebrafish larvae xenografts have enough resolution to display differential tumor responses to Bev in TNBC models and zPDX in just 4 days. We propose zebrafish cancer xenografts as a promising fast in vivo platform for Bev screening, allowing analysis of personalized responses. Citation Format: Cátia Almeida, Raquel Mendes, Joana Mourato Ribeiro, Maria João Cardoso, Fátima Cardoso, Miguel Godinho Ferreira, Rita Fior. Zebrafish xenografts as a fast screening platform for bevacizumab impact on tumor growth, angiogenesis and micrometastasis in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-03-04.

Background: Obstructive Sleep Apnea (OSA) is a highly prevalent sleep disorder associated with multiple comorbidities. Recent studies show that OSA disrupts the biological clock, which might contribute to or play a causal role in the large spectrum of comorbidities observed. Yet, the interplay between OSA, the clock, and OSA treatment is not fully understood. Aim: We explored the impact of short- (4 months) and long-term (24 months) Continuous Positive Airway Pressure (CPAP) treatment on clock-characteristics in OSA patients relative to control subjects. Methods: Plasma melatonin and cortisol, body temperature and the expression levels and rhythmicity of core-clock genes in peripheral blood mononuclear cells (PBMCs) were evaluated, at four time points within 24 h, in 34 adult male OSA patients and 7 sex- and age-matched control subjects. Machine-learning methods were applied for a detailed data analysis. Results: Short- and long-term CPAP promoted distinct alterations in the expression of clock genes in PBMC samples, but none could fully revert OSA-associated alterations. Yet, long-term CPAP re-established the levels and profiles of melatonin, cortisol and body temperature. Clustering approaches based on clock-characteristics could discriminate controls from untreated OSA patients and showed that OSA patients following long-term CPAP better resemble the control than the untreated/short-term treated patients. Conclusion: Long-term CPAP treatment partially ameliorated OSA-associated clock disruptions. A deeper understanding of how OSA and OSA treatment affect the biological clock may open new avenues in the monitoring and treatment of OSA patients.

The role of telomerase reverse transcriptase has been widely investigated in the contexts of ageing and age-related diseases. Interestingly, decreased telomerase activities (and accelerated telomere shortening) have also been reported in patients with emotion-related disorders, opening the possibility for subjective appraisal of stressful stimuli playing a key role in stress-driven telomere shortening. In fact, patients showing a pessimistic judgement bias have shorter telomeres. However, in humans the evidence for this is correlational and the causal directionality between pessimism and telomere shortening has not been established experimentally yet. We have developed and validated a judgement bias experimental paradigm to measure subjective evaluations of ambiguous stimuli in zebrafish. This behavioural assay allows classification of individuals in an optimistic–pessimistic dimension (i.e. from individuals that consistently evaluate ambiguous stimuli as negative to others that perceive them as positive). Using this behavioural paradigm we found that telomerase-deficient zebrafish ( tert − / − ) were more pessimistic in response to ambiguous stimuli than wild-type zebrafish. The fact that individuals with constitutive shorter telomeres have pessimistic behaviours demonstrates for the first time in a vertebrate model a genetic basis of judgement bias.

Tumor models allowing for the in vivo investigation of molecular mechanisms driving tumor progression and metastasis are important to develop novel strategies for cancer treatment. Unfortunately, for Ewing sarcoma no adequate genetic animal models are currently available. Mouse xenograft models are the state of the art to model Ewing sarcoma in vivo. Here, we describe an alternative Ewing sarcoma xenograft model in embryonic and larval zebrafish. This xenograft model offers live imaging and easy compound testing opportunities hereby complementing mouse xenograft models. In this chapter, we provide a detailed protocol how to xenograft Ewing sarcoma cells (shSK-E17T) into 2-day-old zebrafish and how xenografted zebrafish can be imaged and analyzed over consecutive days to study tumor proliferation.

Telomere length is correlated positively with longevity at the individual level, but negatively when compared across species. Here, we tested the association between lifespan and telomere length in African annual killifish. We analyzed telomere length in 18 Nothobranchius strains derived from diverse habitats and measured the laboratory lifespan of 14 strains of N. furzeri and N. kadleci. We found that males had shorter telomeres than females. The longest telomeres were recorded in strains derived from dry region where male lifespans were shortest. At the individual level, we detected a weak negative association between rapid juvenile growth and shorter telomeres in early adulthood. Overall, average telomere length was a good descriptor of telomere length distribution. However, within-individual telomere length spread was not related to any pattern. This substantial variation in telomere length between strains from different environments provides killifish as powerful tool to understand the evolutionarily adaptive value of telomere length.

International audience; Poly (ADP-ribose) polymerase (PARP) inhibition in BRCA-mutated cells results in an incapacity to repair DNA damage, leading to cell death caused by synthetic lethality. Within the treatment options for advanced triple negative breast cancer, the PARP inhibitor olaparib is only given to patients with BRCA1/2 mutations. However, these patients may show resistance to this drug and BRCA1/2 wild-type tumors can show a striking sensitivity, making BRCA status a poor biomarker for treatment choice. Aiming to investigate if the zebrafish model can discriminate sensitivities to olaparib, we developed zebrafish xenografts with different BRCA status and measured tumor response to treatment, as well as its impact on angiogenesis and metastasis. When challenged with olaparib, xenografts revealed sensitivity phenotypes independent of BRCA. Moreover, its combination with ionizing radiation increased the cytotoxic effects, showing potential as a combinatorial regimen. In conclusion, we show that the zebrafish xenograft model may be used as a sensitivity profiling platform for olaparib in monotherapy or in combinatorial regimens. Hence, this model presents as a promising option for the future establishment of patient-derived xenografts for personalized medicine approaches beyond BRCA status.

International audience; Cancer incidence increases exponentially with age when human telomeres are shorter. Similarly, telomerase reverse transcriptase (tert) mutant zebrafish have premature short telomeres and anticipate cancer incidence to younger ages. However, because short telomeres constitute a road block to cell proliferation, telomere shortening is currently viewed as a tumor suppressor mechanism and should protect from cancer. This conundrum is not fully understood. In our current study, we report that telomere shortening promotes cancer in a noncell autonomous manner. Using zebrafish chimeras, we show increased incidence of invasive melanoma when wild-type (WT) tumors are generated in tert mutant zebrafish. Tissues adjacent to melanoma lesions (skin) and distant organs (intestine) in tert mutants exhibited higher levels of senescence and inflammation. In addition, we transferred second generation (G2) tert blastula cells into WT to produce embryo chimeras. Cells with very short telomeres induced increased tumor necrosis factor1-α (TNF1-α) expression and senescence in larval tissues in a noncell autonomous manner, creating an inflammatory environment. Considering that inflammation is protumorigenic, we transplanted melanoma-derived cells into G2 tert zebrafish embryos and observed that tissue environment with short telomeres leads to increased tumor development. To test if inflammation was necessary for this effect, we treated melanoma transplants with nonsteroid anti-inflammatory drugs and show that higher melanoma dissemination can be averted. Thus, apart from the cell autonomous role of short telomeres in contributing to genome instability, we propose that telomere shortening with age causes systemic chronic inflammation leading to increased tumor incidence.

Malfunctions of circadian clock trigger abnormal cellular processes and influence tumorigenesis. Using an in vitro and in vivo xenograft model, we show that circadian clock disruption via the downregulation of the core-clock genes BMAL1, PER2, and NR1D1 impacts the circadian phenotype of MYC, WEE1, and TP53, and affects proliferation, apoptosis, and cell migration. In particular, both our in vitro and in vivo results suggest an impairment of cell motility and a reduction in micrometastasis formation upon knockdown of NR1D1, accompanied by altered expression levels of SNAI1 and CD44. Interestingly we show that differential proliferation and reduced tumour growth in vivo may be due to the additional influence of the host-clock and/or to the 3D tumour architecture. Our results raise new questions concerning host–tumour interaction and show that core-clock genes are involved in key cancer properties, including the regulation of cell migration and invasion by NR1D1 in zebrafish xenografts.

Progressive telomere shortening during lifespan is associated with increased genome instability, block to cell proliferation and aging. Apoptosis and senescence are the two main cellular outcomes upon irreversible cell damage. In this study, we show a transition between apoptosis to senescence in cells of two independent tissues in telomerase zebrafish mutants. In young mutants, proliferative tissues exhibit defects in cell proliferation and p53-dependent apoptosis, but no senescence. Progressively, these tissues display signs of tissue dysfunction, loss of cellularity and increased senescence. These alterations are accompanied by an activation of pro-proliferative stimulus mediated by AKT. Consequently, FoxO1 and FoxO4 transcriptional factors are inactivated, reducing SOD2 levels, causing an increase in ROS. These alterations elicit the activation of the zebrafish p16/15 and senescence. Thus, upon telomere shortening in aging, early apoptosis induces compensatory proliferation. However, progressive decline in cell proliferation results in tissue damage and proliferative signals, promoting a switch to senescence.

International audience; BACKGROUND & AIMS:Vascular invasion is a major prognostic factor in hepatocellular carcinoma (HCC). We previously identified histone H4 acetylated at lysine 16 (H4K16ac), a histone modification involved in transcription activation, as a biomarker of microvascular invasion (mVI) in HCC. This study aimed to investigate the role of hMOF, the histone acetyltransferase responsible for H4K16 acetylation, in the process of vascular invasion in HCC.METHODS:hMOF expression was assessed by RT-qPCR and immunohistochemistry in a retrospective series of HCC surgical samples, and correlated with the presence of mVI. The functional role of hMOF in HCC vascular invasion was investigated in vitro in HCC cell lines using siRNA, transcriptomic analysis and transwell invasion assay, and in vivo using a Zebrafish embryo xenograft model.RESULTS:We found that hMOF was significantly upregulated at the protein level in HCC with mVI, compared with HCC without mVI (P < .01). Transcriptomic analysis showed that hMOF downregulation in HCC cell line lead to significant downregulation of key genes and pathways involved in vascular invasion. These results were confirmed by transwell invasion assay, where hMOF downregulation significantly reduced HCC cells invasion. Finally, hMOF downregulation significantly reduced tumour cell intravasation and metastasis in vivo.CONCLUSIONS:Altogether, these results underpin a critical role for hMOF in vascular invasion in HCC, via transcription activation of key genes involved in this process. These data confirm the major role of epigenetic alterations in HCC progression, and pave the way for future therapies targeting hMOF in HCC.

Cancer incidence increases exponentially with age, when human telomeres are shorter. Similarly, telomerase mutant zebrafish (tert) have premature short telomeres and anticipate cancer incidence to younger ages. However, because short telomeres constitute a road block to cell proliferation, telomere shortening is currently viewed as a tumor suppressor mechanism and should protect from cancer. This conundrum is not fully understood. In our current study, we report that telomere shortening promotes cancer in a non-cell autonomous manner. Using zebrafish chimeras, we show increased incidence of invasive melanoma when WT tumors are generated in tert mutant zebrafish. tert zebrafish show increased levels of senescence (cdkn2a and ink4a/b) and inflammation (TNF-α). In addition, we transferred second generation tert blastula cells into WT to produce embryo chimeras. Cells with very short telomeres induced senescence and increased neutrophil numbers in surrounding larval tissues in a non-cell autonomous manner, creating an inflammatory environment. Considering that inflammation is pro-tumorigenic, we transplanted melanoma-derived cells into second generation tert zebrafish embryos and observed that tissue environment with short telomeres leads to increased micrometastasis. To test if inflammation was necessary for this effect, we treated melanoma transplants with non-steroid anti-inflammatory drugs and show that higher melanoma invasiveness can be averted. Thus, apart from the cell autonomous role of short telomeres in contributing to genome instability, we propose that telomere shortening with age causes systemic chronic inflammation leading to increased tumor incidence.Significance StatementCancer incidence increases exponentially in human midlife. Even though mutation accumulation in somatic tissues results in increased tumorigenesis, it is currently not understood how aging contributes to cancer. Telomeres, the ends of eukaryotic linear chromosomes, shorten with each cell division. Here we show that telomere shortening contributes to cancer in a non-cell autonomous manner. Using embryo chimeras of telomerase deficient zebrafish generated from melanoma-prone fish, we show that tumors arise more frequently and become more invasive in animals with shorter telomeres. Telomere shortening gives rise to increased senescence and systemic inflammation. We observed increased melanoma metastasis dissemination in zebrafish larvae with very short telomeres. Thus, telomere shortening similar to human aging, generates a chronic inflammatory environment that increases cancer incidence.

Background: Whereas the role of neoadjuvant radiotherapy in rectal cancer is well-established, the ability to discriminate between radioresistant and radiosensitive tumors before starting treatment is still a crucial unmet need. Here we aimed to develop an in vivo test to directly challenge living cancer cells to radiotherapy, using zebrafish xenografts. Methods: We generated zebrafish xenografts using colorectal cancer cell lines and patient biopsies without in vitro passaging, and developed a fast radiotherapy protocol consisting of a single dose of 25 Gy. As readouts of the impact of radiotherapy we analyzed proliferation, apoptosis, tumor size and DNA damage. Findings: By directly comparing isogenic cells that only differ in the KRASG13D allele, we show that it is possible to distinguish radiosensitive from radioresistant tumors in zebrafish xenografts, even in polyclonal tumors, in just 4 days. Most importantly, we performed proof-of-concept experiments using primary rectum biopsies, where clinical response to neoadjuvant chemoradiotherapy correlates with induction of apoptosis in their matching zebrafish Patient-Derived Xenografts-Avatars. Interpretation: Our work opens the possibility to predict tumor responses to radiotherapy using the zebrafish Avatar model, sparing valuable therapeutic time and unnecessary toxicity. Keywords: Chemoradiotherapy, Colorectal cancer, Zebrafish avatars, Personalized medicine, Radiotherapy

International audience; Telomeres, the protective ends of eukaryotic chromosomes, are replicated through concerted actions of conventional DNA poly-merases and elongated by telomerase, but the regulation of this process is not fully understood. Telomere replication requires (Ctc1/ Cdc13)-Stn1-Ten1, a telomeric ssDNA-binding complex homologous to RPA. Here, we show that the evolutionarily conserved phos-phatase Ssu72 is responsible for terminating the cycle of telomere replication in fission yeast. Ssu72 controls the recruitment of Stn1 to telomeres by regulating Stn1 phosphorylation at Ser74, a residue located within its conserved OB-fold domain. Consequently, ssu72Δ mutants are defective in telomere replication and exhibit long 3 0-ssDNA overhangs, indicative of defective lagging-strand DNA synthesis. We also show that hSSU72 regulates telomerase activation in human cells by controlling recruitment of hSTN1 to telomeres. These results reveal a previously unknown yet conserved role for the phosphatase SSU72, whereby this enzyme controls telomere homeostasis by activating lagging-strand DNA synthesis, thus terminating the cycle of telomere replication.

Besides its canonical role in stabilizing telomeres, telomerase reverse transcriptase (TERT) may promote tumor growth/progression through extra-telomeric functions. Our previous in vitro studies demonstrated that short-term TERT inhibition by BIBR1532 (BIBR), an inhibitor of TERT catalytic activity, negatively impacts cell proliferation and viability via telomeres&rsquo, length-independent mechanism. Here we evaluate the anti-proliferative and pro-apoptotic effects of short-term telomerase inhibition in vivo in wild-type (wt) and tert mutant (terthu3430/hu3430, tert&minus, /&minus, ) zebrafish embryos, and in malignant human B cells xenografted in casper zebrafish embryos. Short-term Tert inhibition by BIBR in wt embryos reduced cell proliferation, induced an accumulation of cells in S-phase and ultimately led to apoptosis associated with the activation of DNA damage response, all these effects were unrelated to telomere shortening/dysfunction. BIBR treatment showed no effects in tert&minus, embryos. Xenografted untreated malignant B cells proliferated in zebrafish embryos, while BIBR pretreated cells constantly decreased and were significantly less than those in the controls from 24 to up to 72 h after xenotransplantation. Additionally, xenografted tumor cells, treated with BIBR prior- or post-transplantation, displayed a significant higher apoptotic rate compared to untreated control cells. In conclusion, our data demonstrate that short-term telomerase inhibition impairs proliferation and viability in vivo and in human malignant B cells xenografted in zebrafish, thus supporting therapeutic applications of TERT inhibitors in human malignancies.

Aneuploidy, an abnormal chromosome number, has been linked to aging and age-associated diseases, but the underlying molecular mechanisms remain unknown. Here we show, through direct live-cell imaging of young, middle-aged, and old-aged primary human dermal fibroblasts, that aneuploidy increases with aging due to general dysfunction of the mitotic machinery. Increased chromosome mis-segregation in elderly mitotic cells correlates with an early senescence-associated secretory phenotype (SASP) and repression of Forkhead box M1 (FoxM1), the transcription factor that drives G2/M gene expression. FoxM1 induction in elderly and Hutchison–Gilford progeria syndrome fibroblasts prevents aneuploidy and, importantly, ameliorates cellular aging phenotypes. Moreover, we show that senescent fibroblasts isolated from elderly donors’ cultures are often aneuploid, and that aneuploidy is a key trigger into full senescence phenotypes. Based on this feedback loop between cellular aging and aneuploidy, we propose modulation of mitotic efficiency through FoxM1 as a potential strategy against aging and progeria syndromes., Evidence for mitotic decline in aged cells and for aneuploidy-driven progression into full senescence is limited. Here, the authors find that in aged cells, mitotic gene repression leads to increased chromosome mis-segregation and aneuploidy that triggers permanent cell cycle arrest and full senescence.

Creating ways to control drug activation at specific tissues while sparing healthy tissues remains a major challenge. The administration of exogenous triggers offers the possibility for precise and traceless drug activation. However, ensuring localization of the trigger as well as the prodrug at the diseased tissue is complex while essential for therapeutic efficacy and to avoid side-toxicity. Cisplatin remains a first line option to treat 20% of all cancer patients and while clearing after 30 min from blood it concentrates in tumor tissues. Here, we demonstrate the use of the platinum-mediated bond cleavage of protected tertiary amides, which can occur in a catalytic manner under bioorthogonal conditions. Protected analogues of cytotoxic drugs 5-fluorouracil (5-FU) and monomethyl auristatin E (MMAE) were successfully activated using non-toxic amounts of platinum salts in cells. An otherwise fully stable and non-internalizing ADC built using a bifunctional linker featuring a tertiary amide protected MMAE was also bioorthogonally decaged in the presence of platinum salts for extracellular drug release. Finally, cisplatin-mediated activation of a prodrug 5-FU was shown in a colorectal zebrafish xenograft model leading to a significant tumor reduction. Considering cisplatin’s continued use as a first-choice treatment for many solid cancers and especially in colorectal cancer, we anticipate that our platinum-mediated decaging strategy will enhance cancer therapy by allowing tumor specific prodrug activation.

Telomeres, the protective ends of eukaryotic chromosomes, are replicated through concerted actions by conventional DNA polymerases and telomerase, though the regulation of this process is not fully understood. Telomere replication requires (C)-Stn1-Ten1, a telomere ssDNA-binding complex that is homologous to RPA. Here, we show that the evolutionarily conserved phosphatase Ssu72 is responsible for terminating the cycle of telomere replication in fission yeast. Ssu72 controls the recruitment of Stn1 to telomeres by regulating Stn1 phosphorylation at S74, a residue that lies within the conserved OB fold domain. Consequently, ssu72Δ mutants are defective in telomere replication and exhibit long 3’ overhangs, which are indicative of defective lagging strand DNA synthesis. We also show that hSSU72 regulates telomerase activation in human cells by controlling the recruitment of hSTN1 to telomeres. Thus, in this study, we demonstrate a previously unknown yet conserved role for the phosphatase SSU72, whereby this enzyme controls telomere homeostasis by activating lagging strand DNA synthesis, thus terminating the cycle of telomere replication.

The centrosome is composed of two centrioles surrounded by a microtubule-nucleating pericentriolar matrix (PCM). Centrioles regulate matrix assembly. Here we ask whether the matrix also regulates centriole assembly. To define the interaction between the matrix and individual centriole components, we take advantage of a heterologous expression system using fission yeast. Importantly, its centrosome, the spindle pole body (SPB), has matrix but no centrioles. Surprisingly, we observed that the SPB can recruit several animal centriole components. Pcp1/pericentrin, a conserved matrix component that is often upregulated in cancer, recruits a critical centriole constituent, SAS-6. We further show that this novel interaction is conserved and important for centriole biogenesis and elongation in animals. We speculate that the Pcp1/pericentrin-SAS-6 interaction surface was conserved for one billion years of evolution after centriole loss in yeasts, due to its conserved binding to calmodulin. This study reveals an ancestral relationship between pericentrin and the centriole, where both regulate each other assembly, ensuring mutual localisation.

Katsu and Baker (1) highlight a potential weakness in using zebrafish patient-derived xenografts (zPDX) (2) for endocrine-dependent tumors. Katsu and Baker (1) report that, unlike human estrogen receptor (ER), progesterone may activate zebrafish mineralocorticoid receptor (MR) instead of inhibiting it (3). Treatment of zPDXs with progesterone would, therefore, possibly activate zebrafish MR and block human MR, leading to a tumor-autonomous effect that would contrast with a nontumor effect in the host. We would like to highlight the following points regarding their concern. First, zPDXs were primarily designed … [↵][1]1To whom correspondence may be addressed. Email: rita.fior{at}neuro.fchampalimaud.org or miguel.ferreira{at}research.fchampalimaud.org. [1]: #xref-corresp-1-1

Cancer is as unique as the person fighting it. With the exception of a few biomarker-driven therapies, patients go through rounds of trial-and-error approaches to find the best treatment. Using patient-derived cell lines, we show that zebrafish larvae xenotransplants constitute a fast and highly sensitive in vivo model for differential therapy response, with resolution to reveal intratumor functional cancer heterogeneity. We screened international colorectal cancer therapeutic guidelines and determined distinct functional tumor behaviors (proliferation, metastasis, and angiogenesis) and differential sensitivities to standard therapy. We observed a general higher sensitivity to FOLFIRI [5-fluorouracil(FU)+irinotecan+folinic acid] than to FOLFOX (5-FU+oxaliplatin+folinic acid), not only between isogenic tumors but also within the same tumor. We directly compared zebrafish xenografts with mouse xenografts and show that relative sensitivities obtained in zebrafish are maintained in the rodent model. Our data also illustrate how KRAS mutations can provide proliferation advantages in relation to KRASWT and how chemotherapy can unbalance this advantage, selecting for a minor clone resistant to chemotherapy. Zebrafish xenografts provide remarkable resolution to measure Cetuximab sensitivity. Finally, we demonstrate the feasibility of using primary patient samples to generate zebrafish patient-derived xenografts (zPDX) and provide proof-of-concept experiments that compare response to chemotherapy and biological therapies between patients and zPDX. Altogether, our results suggest that zebrafish larvae xenografts constitute a promising fast assay for precision medicine, bridging the gap between genotype and phenotype in an in vivo setting. info:eu-repo/semantics/publishedVersion

The final step of cytoplasmic mRNA degradation proceeds in either a 5′-3′ direction catalysed by Xrn1 or in a 3′-5′ direction catalysed by the exosome. Dis3/Rrp44, an RNase II family protein, is the catalytic subunit of the exosome. In humans, there are three paralogues of this enzyme: DIS3, DIS3L, and DIS3L2. In this work, we identified a novel Schizosaccharomyces pombe exonuclease belonging to the conserved family of human DIS3L2 and plant SOV. Dis3L2 does not interact with the exosome components and localizes in the cytoplasm and in cytoplasmic foci, which are docked to P-bodies. Deletion of dis3l2+ is synthetically lethal with xrn1Δ, while deletion of dis3l2+ in an lsm1Δ background results in the accumulation of transcripts and slower mRNA degradation rates. Accumulated transcripts show enhanced uridylation and in vitro Dis3L2 displays a preference for uridylated substrates. Altogether, our results suggest that in S. pombe, and possibly in most other eukaryotes, Dis3L2 is an important factor in mRNA degradation. Therefore, this novel 3′-5′ RNA decay pathway represents an alternative to degradation by Xrn1 and the exosome.

Telomeres protect the natural ends of chromosomes from being repaired as deleterious DNA breaks. In fission yeast, absence of Taz1 (homologue of human TRF1 and TRF2) renders telomeres vulnerable to DNA repair. During the G1 phase, when non-homologous end joining (NHEJ) is upregulated, taz1Δ cells undergo telomere fusions with consequent loss of viability. Here, we show that disruption of the fission yeast MRN (Rad23MRE11-Rad50-Nbs1) complex prevents NHEJ at telomeres and, as a result, rescues taz1Δ lethality in G1. Neither Tel1ATM activation nor 5′-end resection was required for telomere fusion. Nuclease activity of Rad32MRE11 was also dispensable for NHEJ. Mutants unable to coordinate metal ions required for nuclease activity were proficient in NHEJ repair. In contrast, Rad32MRE11 mutations that affect binding and/or positioning of DNA ends leaving the nuclease function largely unaffected also impaired NHEJ at telomeres and restored the viability of taz1Δ in G1. Consistently, MRN structural integrity but not nuclease function is also required for NHEJ of independent DNA ends in a novel split-molecule plasmid assay. Thus, MRN acts to tether unlinked DNA ends, allowing for efficient NHEJ.

The dramatic telomerase-dependent overelongation of telomeres in cells lacking Taz1 (ortholog of human TRF1/TRF2) or Rap1 implicates these proteins in restraint of telomerase activity. However, the modes by which these proteins regulate telomerase remain mysterious. Here we show that the mechanisms underlying excessive telomerase activity differ markedly between taz1Δ and rap1Δ strains. Despite allowing elevated telomerase access, rap1Δ telomeres are processed and synthesized in a cell-cycle-constrained manner similar to that of wild-type cells. In contrast, taz1Δ telomeres are processed with little cell-cycle dependency and recruit telomerase over an abnormally wide range of cell-cycle stages. Furthermore, although taz1Δ telomeres experience transient attrition mediated by replication fork stalling, this is balanced not only by temporal expansion of the telomerase activity period, but also by markedly increased recruitment of telomerase and its accessory factor Est1, suggesting that stalled forks generate robust substrates for telomerase.